PTEN is one of the most frequently mutated genes in human cancer and the PTEN protein is considered to be a guardian of the genome because of its important role in protecting chromosomal integrity. Observation of structural chromosome aberrations and aneuploidy in cells and tissues lacking functional PTEN brings the link between PTEN and chromosome stability sharply into focus. In our recent studies, we found that cells lacking PTEN exhibit chromosome missegregation and prominent polyploidy, suggesting that PTEN functions during mitosis to control genomic and karyotypic stability. Chromosome instability acquired during aberrant mitosis plays a causative role in tumor development and progression. The long-term goal of this research project is to establish the novel concept that PTEN plays an essential part in controlling mitotic chromosome stability and that its function in maintaining mitotic fidelity is a major driving force in tumor suppression. In support of this aim, our exciting new preliminary data reveal that PTEN deficiency leads to gross genomic alterations manifested by polyploidization and confers cellular resistance to spindle perturbation. Significantly, we found that the protein phosphatase activity of PTEN is required for maintenance of a normal karyotype, and for cellular sensitivity to spindle drugs. In addition, our data have revealed that a key mitotic kinase, polo-like kinase 1 (Plk1), is a potential PTEN target. These findings lead to our hypothesis that PTEN is required for ensuring faithful chromosome inheritance during mitosis and that the protein phosphatase function of PTEN is critical for restraining aberrant mitotic kinase activity in order to maintain a normal karyotype. To test this hypothesis, we propose two specific aims as follows. In Aim 1, we will demonstrate mitotic chromosome instability is a prevailing phenotypic consequence of PTEN deficiency. We will use a Pten conditional knockout leukemia animal model and a panel of Cowden syndrome-derived human lymphoblastoid cell lines to validate the potential causative relationship between PTEN deficiency and karyotypic alterations. In Aim 2, we will define the inhibitory signaling link between PTEN and Plk1 and elucidate the mechanism by which their balanced interplay contributes to karyotypic fidelity and tumor suppression. Using cellular and molecular biology approaches, we will characterize PTEN as an essential mitotic phosphatase and demonstrate that Plk1 is a protein target of the PTEN phosphatase that maintains mitotic chromosome stability. Successful completion of these aims will provide insight into the mechanisms whereby PTEN deficiency triggers mitotic defects and chromosomal instability, and thus leads to tumorigenesis. New findings from this project will offer a new perspective for further mechanistic studies and future development of therapeutic strategies in treating human cancers.